Micro-dissection method for exogenous chromosomes in trititrigia alien substitution line

Abstract

A micro-dissection method for exogenous chromosomes in trititrigia alien substitution line includes: 1) preparing chromosome specimens of root cells of wheat; 2) preparing chromosome specimens of pollen mother cells; 3) performing microscopic dissection of chromosomes of root cells of the wheat; 4) performing microscopic cutting of chromosomes of pollen mother cells; and 5) performing DOP-PCR (degenerate oligonucleotide primed-polymerase chain reaction) on the cut chromosomes of root cells of the wheat and chromosomes of pollen mother cells, detecting a PCR product I and a PCR product II by polyacrylamide native gel electrophoresis, determining that the PCR product II contains target genes of cut common wheat chromosomes in gel imaging system, and determining that the PCR product IV contains target genes of cut intermediate trititrigia exogenous chromosomes in the gel imaging system. The preparation method is simple, the identification method is simple, simple in analyst and convenient in cutting and recovering of the chromosomes and creates basis for subsequent identification and analyst of the cut chromosomes.

Description

technical field [0001] The invention relates to a method for cutting and identifying exogenous chromosomes, in particular to a method for micro-separating exogenous chromosomes in wheat-Thinopyrum intermedium heterosubstitution lines, belonging to the field of molecular cytogenetics. Background technique [0002] Chromosome microdissection and microcloning (chromosome microdissection and microcloning) technology is a new technology of molecular cytogenetics founded by Scalenghe in 1981. The technology first cuts the Drosophila chromosome, and successfully obtained 80 clones, which were then used to cut the chromosomes of mice and humans. With the development of PCR technology, micro-dissection and micro-cloning technology has been greatly developed. Since the use of chromosome micro-dissection and micro-cloning technology to obtain rye DNA clones, this technology has been used in the construction of plant chromosome libraries, specific probe screening, and anti-bacteria. Th...

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Problems solved by technology

[0003] (1) The existence of plant cell walls and the difficulty of synchronization hinder the preparation of well-distributed chromosome specimens and affect the cutting efficiency

[0004] (2) The ploidy of the plant genome is diverse, the chromosome structure varies greatly, and the sequence of genes is not conservative enough; the DNA content of plant chromosomes is high and the protein content is low, and most chromosomes do not show G, R or Q bands, which affects the DNA sequence. The prevailing C-band damages the chromosomal DNA too much, which is not conducive to the construction of a complete DNA library; while some plants have no obvious difference in chromosome size and similar band patterns, it is difficult to accurately identify chromosomes and their fine structures, which increases the complexity of analysis

[0005] (3) There are many highly repetitive sequences in plant chromosomal DNA, such as about 80% of the repetitive sequences in the nuclear genomes of echinopsis, barley, rye and oats, and the DNA content of wheat crops is high, such as the DNA content of hexaploid oats is 13.2-14.3 pg, the C value of Thiopyrum intermedia is 1.7×10 9 bp, an average of 0.8×10 per chromosome 9 bp, equivalent to a quarter of the human genome If each insert is 1kb, each chromosome-specific DNA library contains 8×10 5 For the above clones, the workload of screening library is huge

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